Apolipoprotein (apo) E4 is the major genetic risk factor for Alzheimer?s disease (AD). In fact, more than 70 million Americans are at higher risk for AD because of their apoE4 carrier status. Two-thirds of AD patients are apoE4 carriers, with apoE4 increasing the risk and decreasing the age of onset of this devastating disease. Understanding how apoE4 causes neuropathology is critically important because it will guide the development of therapies to retard, or possibly prevent, apoE4-associated neuropathology. This proposal builds on our hypothesis that apoE4-associated neuropathology is related to the susceptibility of apoE4 to neuron-specific proteolysis. Neurotoxic fragments resulting from the proteolysis escape the secretory pathway and enter the cytosol where they alter several cellular and metabolic processes (e.g., mitochondrial dysfunction and cytoskeletal alterations). Human apoE4 carriers, even 20?30-year-old cognitively normal subjects, display brain glucose hypometabolism and impaired mitochondrial enzyme activity. In mouse neurons expressing human apoE4, several abnormalities arise. For example, apoE4 is associated with impaired mitochondrial respiration, neurite outgrowth and neurotoxicity, and these can be reversed by apoE4 structure correctors that convert apoE4 to an apoE3-like conformation, thus preventing neurotoxic fragment generation and neurodegeneration. Despite our advances in understanding the genesis and ultimate consequences of apoE4 proteolysis in neurons, many of the defining intermediate steps remain unclear. Indeed, our preliminary results indicate potentially broad detrimental downstream effects of apoE4 expression in neural cells. The goal of this project is to exploit the combined expertise of the Mahley and Krogan laboratories and advanced proteomic and genetic analyses of our models of apoE4-mediated neurotoxicity to establish how apoE4 alters neuronal processes/metabolism. We will combine label-free quantitative mass spectrometry?based protein-protein interaction pathway analysis with profiling of the total and post-translational modification proteomes, and validate targets using transcriptomic profiling and the latest CRISPRi/a gene regulation technologies. Comparing changes in neuronal protein networks associated with the expression of apoE3, apoE4, and the predominant apoE4(1?272) fragment in cultured neurons will elucidate the critical mediators of apoE4 neurotoxicity. Treatment of the apoE4 neurons with an apoE4 structure corrector will establish the importance of apoE4 structure to alterations in the neuronal proteome. Our joint expertise positions us well to address these longstanding questions about apoE4 function in the nervous system, and our preliminary data demonstrate the validity of our methods to identify new targets. Our multi-omic approach will yield mechanistic insights into apoE4-driven neuropathology and fill critical gaps in our understanding of AD pathogenesis.